HPV16 and BPV1 infection can be blocked by the dynamin inhibitor dynasore - PubMed (original) (raw)

HPV16 and BPV1 infection can be blocked by the dynamin inhibitor dynasore

Cynthia Y Abban et al. Am J Ther. 2008 Jul-Aug.

Abstract

The initial entry of papillomaviruses into their target cells has been shown to occur by clathrin-mediated endocytosis and caveolae-mediated endocytosis. These mechanisms entail the formation of nascent-coated vesicles at the plasma membrane. Such coated vesicles, clathrin or caveolin, form and pinch-off in a controlled mechanism that involves several proteins including dynamin. Dynamin is a GTPase that forms a dynamin ring at the stem connecting the nascent vesicle to the plasma membrane. In a still not fully characterized mechanism, dynamin's contraction and twisting results in the scission of the vesicle. In an effort to better characterize the role and molecular mechanisms of dynamin's function, researchers have identified dynasore, a dynamin GTPase inhibitor that prevents the scission of dynamin-dependent endocytic vesicles. Here, we have tested if infection by pseudovirus corresponding to the oncogenic human papillomavirus type 16 and bovine papillomavirus type 1 can be blocked by dynasore. We present data demonstrating that dynasore can block infection of human papillomavirus type 16 and bovine papillomavirus type 1 pseudovirions in a dose- and time-dependent manner with equal efficiency. Presently, there is no available therapy that can block infection by a wide range of papillomavirus regardless of species or genotypes. Targeting dynamin may lead to the rational design of drug able to prevent infection by papillomaviruses, and by other infectious agents dependent on this protein for initial internalization into target cells. Whether such an approach will prove successful needs further investigation.

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Figures

FIGURE 1

FIGURE 1

Dose-dependent inhibition of PV infection by dynasore inhibitor. HEK 293 cells were infected with BPV1 or HPV16 pseudovirions carrying the GFP cDNA, in the presence of 0, 20, 40, 80, and 100 μM dynasore. Infection was measured by FACS analysis of GFP-positive cells. Line with diamond is the BPV1 dose–response curve, and line with the rectangle is the HPV16 dose–response curve.

FIGURE 2

FIGURE 2

Dynasore inhibition of viral infection depends on the time of addition of the inhibitor. Dynasore (80 μM/mL) was added to 293 cells at different times during infection, and percent inhibition was measured by FACS analysis of GFP-positive cells. Bars: −30 minutes, dynasore was added 30 minutes before adding the virus; 0 minutes, dynasore was added just before incubating cells at 37°C; +30 minutes, dynasore was added 30 minutes after incubation at 37°C; +2 hours, dynasore was added 2 hours after infection; +4 hours, dynasore was added 4 hours after infection. Each bar is representative of independent experiments carried out in triplicates.

FIGURE 3

FIGURE 3

The inhibitory effect of dynasore is partly reversible. (A) Control panel: HEK 293 cells incubated with alexa-fluor 594–labeled transferrin; dynasore panel: cells were incubated with 80 μM/mL dynasore for 30 minutes and then labeled transferrin was added to media; washout panel: cells were incubated with 80 μM/mL dynasore for 30 minutes, media were replaced with dynasore-free fresh media, and labeled transferrin was added after 20 minutes. All steps were incubated at 37°C. Remaining surface transferrin was removed by acid wash before fixation and imaging of cells. Transferrin in red, and nuclei in blue (DAPI). (B andC)Black bars: percent infection of cells incubated with DMSO for 30 minutes and infected with BPV1 in B, HPV16 in C virus; gray bars: percent infection of cells incubated with dynasore for 30 minutes and infected with BPV1 virus; white bars: percent infection of cells incubated with dynasore for 30 minutes and infected with BPV1 virus. Dynasore was washed off (white bars) at −30 minutes, 0 hour, +30 minutes, +2 hours, and +4 hours, in relation addition of virus.

FIGURE 4

FIGURE 4

Electron microscopic analysis of the effect of dynasore inhibitor. (A) HEK 293 cells incubated for 30 minutes with 80 μM dynasore. (B–E) HEK 293 cells infected with BPV1 pseudovirus for 1 hour in the absence of dynasore. (F–I) HEK 293 cells infected with BPV1 pseudovirus for 1 hour in the presence of 80 μM dynasore. Scale bar represents 100 nm; ×80,000 magnification.

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